WO1999027955A2 - Traitement d'infections chroniques etablies et de cancers - Google Patents

Traitement d'infections chroniques etablies et de cancers Download PDF

Info

Publication number
WO1999027955A2
WO1999027955A2 PCT/GB1998/003583 GB9803583W WO9927955A2 WO 1999027955 A2 WO1999027955 A2 WO 1999027955A2 GB 9803583 W GB9803583 W GB 9803583W WO 9927955 A2 WO9927955 A2 WO 9927955A2
Authority
WO
WIPO (PCT)
Prior art keywords
dendritic cells
substance
cytokine
cells
hel
Prior art date
Application number
PCT/GB1998/003583
Other languages
English (en)
Other versions
WO1999027955A3 (fr
Inventor
Benjamin Michael Chain
Peter Charles Leonard Beverley
Alexander Hal Drakesmith
Deborah Antoinette O'neil
Original Assignee
University College London
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by University College London filed Critical University College London
Priority to AU13432/99A priority Critical patent/AU1343299A/en
Publication of WO1999027955A2 publication Critical patent/WO1999027955A2/fr
Publication of WO1999027955A3 publication Critical patent/WO1999027955A3/fr

Links

Classifications

    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K2239/00—Indexing codes associated with cellular immunotherapy of group A61K39/46
    • A61K2239/38—Indexing codes associated with cellular immunotherapy of group A61K39/46 characterised by the dose, timing or administration schedule
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • A61K38/19—Cytokines; Lymphokines; Interferons
    • A61K38/20—Interleukins [IL]
    • A61K38/204—IL-6
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • A61K38/19—Cytokines; Lymphokines; Interferons
    • A61K38/21—Interferons [IFN]
    • A61K38/217—IFN-gamma
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/46—Cellular immunotherapy
    • A61K39/461—Cellular immunotherapy characterised by the cell type used
    • A61K39/4615—Dendritic cells
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/46—Cellular immunotherapy
    • A61K39/462—Cellular immunotherapy characterized by the effect or the function of the cells
    • A61K39/4621—Cellular immunotherapy characterized by the effect or the function of the cells immunosuppressive or immunotolerising
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/46—Cellular immunotherapy
    • A61K39/462—Cellular immunotherapy characterized by the effect or the function of the cells
    • A61K39/4622—Antigen presenting cells
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/46—Cellular immunotherapy
    • A61K39/464—Cellular immunotherapy characterised by the antigen targeted or presented
    • A61K39/4643—Vertebrate antigens
    • A61K39/46433—Antigens related to auto-immune diseases; Preparations to induce self-tolerance
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/06—Animal cells or tissues; Human cells or tissues
    • C12N5/0602—Vertebrate cells
    • C12N5/0634—Cells from the blood or the immune system
    • C12N5/0639—Dendritic cells, e.g. Langherhans cells in the epidermis
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K48/00—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2501/00—Active agents used in cell culture processes, e.g. differentation
    • C12N2501/20—Cytokines; Chemokines
    • C12N2501/23—Interleukins [IL]
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2501/00—Active agents used in cell culture processes, e.g. differentation
    • C12N2501/999—Small molecules not provided for elsewhere

Definitions

  • the present invention relates to the treatment of established chronic infections and cancers. More particularly, but not exclusively, it relates to cytokines and their use in such treatment and the use of cytokines to modify the immune response of dendritic cells in the treatment of such conditions.
  • an "established chronic infection” is an infection to which an immune response has been raised by the infected patient but which has not been cleared or neutralised by that immune response.
  • infections are infection by HIV, Hepatitis B Virus (HBV) , Herpes Simplex Virus (HSV) , malaria and Mycobacterium tb.
  • Dendritic cells are key cells required for the initiation of many immunological responses. Specifically, their activity is required to activate the T lymphocyte which regulates immunological responses to antigens. An essential part of this activity is the ability of dendritic cells to cut up protein antigens into smaller fragments, known as epitopes, and to allow these epitopes to bind to molecules of the major histocompatibility complex (MHC) , both class I and class II. In practice, the dendritic cell only allows a very small proportion of epitopes to bind to these MHC molecules, and hence the immune response is restricted to a small number of such epitopes .
  • MHC major histocompatibility complex
  • the inventors have found that treatment of dendritic cells with a substance which causes acidification of the early endosome of such cells, causes these cells to present cryptic and subdominant epitopes at higher levels than they would otherwise be presented.
  • This "broadening" of the range or repertoire of epitopes that are presented by dendritic cells means that substances which cause acidification of the early endosome of dendritic cells can be used to provoke an immune response against epitopes to which the body has not yet become tolarised.
  • this "broadening" of the range or repertoire of epitopes that are presented by dendritic cells can be caused by treating dendritic cells with cytokines, in particular IL-6, and analogues thereof, thereby causing the acidification of the early endosome .
  • analogue used herein is intended to include active parts of cytokines, molecules which mimic the activity of cytokines and active parts of such molecules.
  • cytokine refers to both cytokines and analogues thereof .
  • a pharmaceutically-acceptable substance which causes acidification of the early endosome of dendritic cells, in the production of a medicament for the treatment of an established chronic infection or a cancer.
  • the substance is an inhibitor of the Na + /K + ATPase of dendritic cells and may be, for example , ouabain .
  • the substance may be a cytokine or a DNA molecule coding for a cytokine .
  • the cytokine or DNA molecule coding for a cytokine preferably upregulates the presentation of the 2-16 epitope of hen egg lysozyme (HEL) by dendritic cells in the assay defined herein.
  • HEL hen egg lysozyme
  • Cytokines useful in the present invention may have the property that they upregulate the presentation of the 2-16 epitope of HEL in an assay having the following conditions : Dendritic cells are grown from BALB/c or CBA female 8-12 week old mouse bone marrow precursor cells (in Iscove's Modified Dulbecco's Medium (Gibco BRL) supplemented with FCS and 5 x 10 "5 2-mercaptoethanol (2-ME) ) for 6 days in the presence of 10 ng/ml recombinant murine granulocyte-macrophage colony-stimulating factor (GM-CSF, Autogen Bioclear) as described in (ref 3) .
  • GM-CSF murine granulocyte-macrophage colony-stimulating factor
  • the dendritic cells are then cultured for a further 24 or 48 hours, with or without 5 ng/ml of added cytokine at 10 6 cells/ml and 10 ng/ml GM-CSF.
  • the residual cytokine is removed by washing three times, and 10 4 dendritic cells/well are cultured (in RPMl/5% FCS/2-ME) for 24 hours with varying doses (1- 100 ⁇ g/ml) of whole HEL (Boehringer Mannheim) and 5 x 10 4 cells of a T cell line specific for the 2-16 epitope .
  • the ability of the 2-16 epitope to stimulate the T cell is measured by release of IL-2 after 24 hour culture as described in (ref 12) .
  • IL-2 The ability of the 2-16 epitope to stimulate the T cell is measured by release of IL-2 after 24 hour culture as described in (ref 12) .
  • supernatants are removed and IL-2 levels are assayed by proliferation of the IL-2 dependent cell line CTLL measured by 3 H-thymidine (Amersham) incorporation .
  • a cytokine upregulates the presentation of the 2- 16 epitope of HEL when the CTLL proliferation (as measured in cpm/l0e3) for dendritic cells treated with cytokine is at least three times the background release of IL-2 for dendritic cells not treated with cytokine.
  • the established chronic infection or cancer can be treated using the substance in a number of methods.
  • dendritic cells can be extracted from the patient, exposed to the substance, and then reintroduced into the patient.
  • the dendritic cells treated in this way will present an increased repertoire of epitopes, increasing the likelihood that an immune response will be raised to one or more antigens associated with the established chronic infection or cancer.
  • the invention provides a vaccine for an established chronic infection or a cancer, comprising dendritic cells exposed to a substance which upregulates the presentation of the 2- 16 epitope of HEL by dendritic cells in the assay defined herein and/or causes acidification of the early endosome of dendritic cells.
  • dendritic cells are also included within the invention.
  • the dendritic cells of the vaccine are preferably also exposed to the specific antigen.
  • dendritic cells extracted from the patient are exposed to the substance and the specific antigen, and reintroduced into the patient.
  • the dendritic cells treated in this way will present an increased repertoire of epitopes of the specific antigen, increasing the likelihood that an immune response will be raised to that antigen.
  • cancers having a known specific antigen are ovarian cancer, lymphoma and melanoma.
  • established chronic infections having a known specific antigen are HIV, Hepatitis B Virus (HBV) , Herpes Simplex Virus (HSV) , malaria and Mycobacterium tb.
  • HIV Hepatitis B Virus
  • HSV Herpes Simplex Virus
  • the substance can be injected, or otherwise introduced into the patient, so that the dendritic cells of the patient are exposed to the substance and antigen (s) associated with the established chronic infection or cancer in vivo .
  • the substance is preferably injected, or otherwise introduced into the patient, together with the specific antigen so that the dendritic cells of the patient are exposed to the substance and specific antigen in vivo .
  • the invention provides a vaccine for an established chronic infection or a cancer, comprising: a substance which upregulates the presentation of the 2-16 epitope of HEL by dendritic cells in the assay as defined herein and/or causes acidification of the early endosome of dendritic cells; and an antigen specific to the established chronic infection or cancer.
  • the dendritic cells can be exposed to the substance using gene therapy, i.e. by introducing into the patient DNA coding for the substance .
  • the invention provides a vaccine for an established chronic infection or a cancer, comprising: a first DNA molecule coding for a substance which upregulates the presentation of the 2-16 epitope of HEL by dendritic cells in the assay as defined herein, and/or causes acidification of the early endosome of dendritic cells.
  • the vaccine when the established chronic infection or cancer has a known specific antigen, the vaccine preferably further comprises a second DNA molecule coding for the antigen specific to the established chronic infection or cancer.
  • the first and second DNA molecules may be introduced in the same or different expression vector or by DNA vaccination techniques.
  • the established chronic infection or cancer can be treated by causing cells of the tissue infected with the established chronic infection or cancer cells to themselves present antigen. This is achieved by transfecting the tissue or cancer cells with DNA coding for one or more co- stimulatory molecules so that the or each co- stimulatory molecule is expressed on the cell surface.
  • the transfected cells are exposed to the substance either by injecting the substance, or preferably by also transfecting the cells with DNA coding for the substance. This method is especially useful where the established chronic infection or cancer does not have a known protective specific antigen.
  • the invention provides a vaccine for an established chronic infection or a cancer, comprising: a first DNA molecule coding for a substance which upregulates the presentation of the 2- 16 epitope of HEL by dendritic cells in the assay as defined herein and/or causes acidification of the early endosome of dendritic cells; and a second DNA molecule coding for a co-stimulatory molecule.
  • first and second DNA molecules are preferably linked.
  • the first and second DNA molecules may be introduced in an expression vector or by DNA vaccination techniques.
  • a method of increasing the range of epitopes of an antigen presented by a dendritic cell comprising: exposing the dendritic cell to (i) the antigen and (ii) a substance which upregulates the presentation of the 2-16 epitope of HEL by dendritic cells in the assay defined herein and/or causes acidification of the early endosome of dendritic cells .
  • This method is preferably used in the treatment of an established chronic infection or a cancer.
  • the substance may be an inhibitor of the Na + / + ATPase of dendritic cells and may be, for example, ouabain.
  • the substance is preferably a cytokine or a DNA molecule coding for a cytokine.
  • the cytokine or DNA molecule coding for a cytokine preferably upregulates the presentation of the 2-16 epitope of HEL by dendritic cells in the assay defined herein.
  • the cytokine may be a pro-inflammatory cytokine.
  • the inventors have found that the cytokines IL-6 and interferon- ⁇ have this property.
  • the cytokine used in the present invention acts on the IL-6 receptor.
  • the cytokine is IL-6.
  • the present invention provides the use of a cytokine blocker, which downregulates the presentation of the 2-16 epitope of HEL in the assay defined herein and/or prevents acidification of the early endosome of dendritic cells, in the production of a medicament for the treatment of an autoimmune disease .
  • cytokine blocker is a molecule which downregulates the presentation of the 2-16 epitope of
  • the blocker may block the action of a cytokine or cytokine analogue which upregulates the presentation of the 2-16 epitope of HEL.
  • Examples of such blockers are antibodies or other agents which bind to cytokines, and antibodies or other agents which bind to cytokine receptors .
  • the cytokine blocker may block the activity of a pro-inflammatory cytokine.
  • the inventors have found that the cytokines IL-6 and IF- ⁇ have the property of upregulating the presentation of the 2-16 epitope of HEL. Accordingly, blockers of these cytokines or their receptors are preferred. Most preferred are blockers of IL-6 or the IL-6 receptor.
  • the invention also includes within its scope DNA molecules coding for cytokine blockers and the use of such DNA molecules in the treatment of autoimmune disease .
  • a method of decreasing the range of epitopes of an antigen presented by a dendritic cell comprising: exposing the dendritic cell to (i) the antigen and (ii) a cytokine blocker which downregulates the presentation of the 2-16 epitope of HEL in the assay defined herein and/or prevents acidification of the early endosome of dendritic cells.
  • This method is preferably used in the treatment of an autoimmune disease.
  • HEL hen egg lysozyme
  • Iscove's Modified Dulbecco's Medium (Gibco BRL) supplemented with FCS/ 5 x 10 "5 2-mercaptoethanol (2 -ME) was used for dendritic cell culture.
  • RPMI/5% FCS/2-ME was used for hybridoma culture and hybridoma assays .
  • HL-1 serum-free medium (Biowhittaker) was used for ex vivo lymphocyte proliferation assays Dendritic cell culture
  • Dendritic cells were grown from BALB/c or CBA female 8-12 week old mouse bone marrow precursor cells for 6 days in the presence of 10 ng/ml recombinant murine granulocyte-macrophage colony- stimulating factor (GM-CSF. Autogen Bioclear) as described (ref 3) . On day 6, non-adherent cells and loosely adherent colonies were removed by gentle pipetting and subcultured overnight at 10 6 cells/ml with 10 ng/ml GM-CSF plus or minus recombinant murine 5 ng/ml IL-6 or 10 ng/ml TNF- ⁇ (Autogen Bioclear) ; in some experiments 20 ⁇ M chloroquine (Sigma) as also added.
  • GM-CSF granulocyte-macrophage colony- stimulating factor
  • non-adherent cells were washed three times and used for hybridoma assays, staining, or immunisation.
  • Non- adherent cells were routinely >80% dendritic cells (MHC class II positive, CDllc positive as revealed with N418 mAb (A. Livingstone, biotinylated by C. Van Wely) ) , with remaining cells being MHC Class II negative granulocytes .
  • Hybridoma assay DC or DC/IL-6 were cultured at 10 4 cells/well with hybridoma cells (ED2 cells available from the inventors) at 5 x 10 4 cells/well with whole HEL (Boehringer Mannheim) or HEL peptide (synthesised at ICRF) at graded doses, all in triplicate. After 24 hours, supernatants were removed and IL-2 levels were assayed by proliferation of the IL-2 dependent cell line CTLL measured by 3 H-thymidine (Amersham) incorporation over the last 18 hours of a 36 hour culture. Neither DC, DC/IL-6 nor any hybridoma alone secreted significant IL-2. Phenotypic analysis
  • CBA DC and DC/IL-6 were labelled with mouse anti- I-A k (ATCC, Tib 93) which was visualised using a second layer goat anti-mouse IgG2b-FITC (Nordic) .
  • Flow cytometric analysis was performed on a Becton-Dickinson FACScan.
  • DC or DC/IL-6 from CBA or BALB/c mice were cultured with whole HEL at 50 ⁇ g/ml for 6 hours, washed three times, resuspended at 10 s cells/ml in PBS and 5 x 10 4 cells/foot were injected into opposite hind footpads of 6 CBA or BALB/c mice.
  • lymphocytes obtained were cultured at 5 x 10 5 cells/well in triplicate with HEL, ovalbumin (Sigma, grade VII) or HEL peptides at 6 ⁇ g/ml for 4 days and proliferation was measured by 3 H-thymidine incorporation over the last 18 hours of culture (ref 4) .
  • HEL (100 ⁇ g/mouse) plus or minus IL-6 (5 ng/mouse) in PBS were emulsified at 1:1 ratio in IFA (Sigma) , then injected at the base of tail into 5 CBA or BALB/c mice. 10 days later, spleens were harvested and splenocytes were cultured at 10 6 cells/well for 5 days with antigens as above. Background values of proliferation without added antigen (-2000 cpm) were subtracted.
  • HEL plus or minus IL-6 as above, or ovalbumin (100 ⁇ g/foot) plus IL-6 were emulsified in IFA as above and injected into opposing rear footpads of 10 BALB/c mice. 5 days later draining nodes were harvested and resulting lymphocytes were depleted of T cells using anti-Thy antibody (Serotec) and rabbit complement (Cedarlane Laboratories) and separated into B cells and N418 positive dendritic cells as described (ref 5) .
  • the dendritic ce] 1 fraction was >85% pure (contaminant were B220 positive B cells) while the B cell fraction contained less than 2% dendritic cells.
  • HEL 2-16 specific hybridoma cells were cultured at graded concentrations with HEL 2-16 specific hybridoma cells at 5 x 10 4 cells/well with or without HEL 2-16 specific hybridoma cells at 5 x 10 4 cells/well with or without HEL 2-16 peptide at 100 ⁇ g/ml for 24 hours.
  • IL-2 secreted by the hybridoma was measured using CTLL cells as above. Location of acidic vesicles using DAMP
  • dendritic cells with and without IL-6 were incubated with 30 ⁇ M DAMP (N-(3-((2,4- dinitrophenyl) amino) propyl) -N- (3-aminopropyl) methylamine dihydrochloride, Molecular Probes) for 30 minutes, then washed, fixed, permeabilised, stained with a mouse anti-DNP antibody (B. Askonas, Imperial
  • Example 1 Six day old bone marrow derived BALB/c (H-2 d ) or CBA (H-2 k ) mouse dendritic cells were subcultured overnight with IL-6 (DC/IL-6) or without added IL-6 (DC) . These cells were then cocultured with whole HEL or an HEL peptide determinant and an epitope-specific T-T cell hybridoma.
  • Figures 2a-1 of the accompanying drawings where the open symbols are for DC/lL-6, and the solid symbols are for DC.
  • the lefthand column shows the results when dendritic cells were cultured with whole HEL and the righthand column shows the results when dendritic cells were cultured with an HEL peptide determinant.
  • Figures 2a-d show the results for CBA mouse dendritic cells and Figures 2e-l show the results for BALB/c mouse dendritic cells.
  • Hybridoma responses are expressed as proliferation of CTLL as the mean of triplicates with all values within 10% of the mean. With zero antigen, hybridoma responses to DC/IL-6 and DC were not significantly different and cpm values were typically less than 2000. It can be seen that, when cocultured with whole HEL, DC/IL-6 elicited a different profile of T-T cell hybridoma responses compared to DC. With I-A restricted determinants, DC/IL-6 presented the codominant determinant 46-61 much less efficiently than DC ( Figure 2a) , while display of the subdominant determinant 74-86 ( Figure 2c) was not significantly affected.
  • DC/IL-6 presented the dominant determinant 106-116 as well as DC ( Figure 2i) .
  • the cryptic determinant 2-16 was efficiently processed and displayed from whole HEL only by DC/IL-6 ( Figure 2k) .
  • DC and DC/IL-6 were able to present the respective peptide form of all determinants to the T-T cell hybridomas equivalently (righthand column) . This shows that both DC and DC/lL-6 could present each determinant if it required minimal processing, but that they processed native HEL differently, displaying two distinct sets of determinants.
  • Figure 3 of the accompanying drawings shows the results of measuring surface levels of MHC class II for DC (a) , DC/IL-6 (b) and TNF-c treated DC for CBA dendritic cells, by staining for MHC class II dimer I- A k .
  • Control indicates staining of cells with 2nd layer (goat anti-mouse Ig FITC) alone. Results of staining BALB/c dendritic cells for surface I-A d and I-E d gave similar results (not shown) .
  • Example 3 In this example, it was investigated whether
  • DC/IL-6 could prime T cells from the in vivo repertoire against cryptic and subdominant HEL determinants .
  • DC and DC/IL-6 were exposed to HEL and were injected into opposite hind footpads of CBA and BALB/c mice. Five days later the recall specificities of draining node lymphocytes for HEL or HEL determinants were tested .
  • T cells from nodes draining DC/IL-6 immunisation sites responded well to N-terminal peptides, namely subdominant determinants 11-25 and 23-32, and particularly the cryptic determinant 2-16. Responses to other determinants were unchanged, and the response to whole HEL was increased relative to cells from nodes draining DC immunisation sites. These in vivo responses are consistent with the in vi tro responses of the T-T hybridomas to DC/IL-6 and DC.
  • T cell proliferative responses to those determinants from mice immunised by whole antigen ref 7
  • dominant determinants elicit the greatest responses (ref 1) .
  • the inventors investigated whether injecting IL-6 with antigen could affect this hierarchy of T cell responses .
  • mice were immunised with HEL plus or minus IL-6 emulsified in Incomplete Freund' s Adjuvant (IFA) at the base of the tail, and ten days later splenocytes were cultured with HEL or HEL determinants. Proliferation was measured for each individual mouse .
  • IFA Incomplete Freund' s Adjuvant
  • HEL/IFA black symbols
  • HEL+IL-6/IFA white symbols
  • Proliferation was measured as shown in Figure 4.
  • Data points represent responses of individual mice.
  • CBA mice Figure 5a
  • BALB/c mice Figure 5b
  • the responses to whole HEL 11-25, 23-32 and most dramatically 2-16 were upregulated. Similar results were obtained using draining node lymphocytes instead of splenocytes (not shown) .
  • the generally improved T cell responses to cryptic and subdominant determinants observed here is reminiscent of the recruitment of diverse T cells found in the epitope spreading phenomenon noted in autoimmune disease progression (refs 4, 8, 9) .
  • tissue dendritic cells present the processed antigen to T cells in lymphoid organs (ref 5) .
  • IL-6 is a growth/differentiation factor for B cells (ref 10)
  • activated B cell may also prime naive T cells (ref- 11)
  • the inventors tested whether B cells or dendritic cells were presenting the cryptic determinant when IL-6 was added to the antigen.
  • mice were injected with HEL in IFA or HEL plus IL-6 in IFA into opposing footpads. Five days later draining node lymphocytes were depleted of T cells, and dendritic cells or B cells were separated from the remaining population before coculture with a T-T hybridoma specific for cryptic determinant HEL 2- 16.
  • FIG. 6a-c of the accompanying drawings circles show the results for B cells and triangles show the results for dendritic cells when incubated with cryptic determinant (HEL2-16) specific hybridoma cells with (black symbols) and without (white symbols) HEL2-16 peptide.
  • Figure 6a is for cells obtained from BALB/c mouse contralateral lymph nodes draining HEL/IFA immunisations, 6b is for HEL+IL-6/IFA immunisations and 6c is for a separate experiment draining ovalbumin+IL-6/IFS immunisations Hybridoma responses are expressed as in Figure 2.
  • IL-6 affected the processing of HEL by dendritic cells, which then displayed the cryptic determinant in the lymph nodes. Therefore, the altered specificities of the T cell responses to cryptic and other epitopes observed in Figure 5 were most likely due to the influence of IL-6 on dendritic cells.
  • the acidotrophic antigen processing inhibitor chloroquine has been used to determine if processing of particular determinants from an antigen requires acidic intracellular compartments .
  • the inventors attempted to use chloroquine to investigate the processing of HEL by IL-6 treated dendritic cells.
  • IL-6 may have a role in heightened vesicle acidification in dendritic cells, so that chloroquine might accumulate in and be toxic to IL-6 treated calls but not to controls.
  • Example 7 The possible acidification of dendritic cell vesicles by IL-6 was investigated further using DAMP as described above.
  • a comparison between dendritic cells and dendritic cells and IL-6 showed that, when IL-6 is added, acidic vesicles appear in peripheral areas of the cytoplasm just below the plasma membrane.
  • Dendritic cells and IL-6 were allowed to endocytose 1 mg/ml Texas-Red conjugated dextran
  • Double-labelling of the "early" Texas-Red positive compartments confirmed that they contained the early endosome marker CD71 (the transferrin receptor) , but were negative for the lysosomal marker LAMP-1 (not shown) .
  • a hybridoma assay was carried out as in Example 1 with an increasing concentration of ouabain.
  • the results are shown in Figure 8 of the accompanying drawings. These results indicate that ouabain allows processing and presentation of HEL 2-16 peptide from whole HEL (closed circles) but does not interfere with presentation of HEL 2-16 peptide (open circles) by dendritic cells to HEL 2-16 specific hybridoma cells. Ouabain did not affect the rate of fluid-phase endocytosis (not shown) . Experiments were carried out three times with similar results.
  • IL-6 treated dendritic cells were much less efficient at stimulating responses against a hitherto codominant determinant, HEL 46-61 in the H-2 k haplotype . In contrast, they acquired the ability to activate T cells against other determinants, including the cryptic determinant HEL 2-16, which is not normally recognized following exposure to intact antigen.
  • a key event in the molecular mechanisms mediating the effect of IL-6 appears to be the increased acidification of peripheral early endosomes by IL-6, which may be mediated via inhibition of the endosomal Na + /K + ATPase.
  • lower pH in the early endosome should favour an exchange of bound MHC class II associated invariant chain peptide (CLIP) for antigenic peptide : mild acid pH favours the association of HLA-DM with the MHC class II/invariant chain complex, the spontaneous or HLA-DM-mediated removal of CLIP from MHC class II, stabilises empty forms of MHC class II generated after removal of CLIP, and enables peptide loading of MHC class II, possibly by inducing a conformational change in the MHC class II dimer. This could allow cryptic determinants revealed by altered coprocessing to be rescued by binding to MHC class II before they are destroyed by the full battery of late endosome proteinases .
  • CIP MHC class II associated invariant chain peptide
  • IL-6 is absolutely essential in several experimental autoimmune encephalomyelitis and arthritis models.
  • high levels of IL-6 may cause dendritic cells to process self antigen qualitatively differently so that previously hidden (cryptic) determinants are revealed to the T cell repertoire.
  • blocking antigen processing by IL-6 exposed dendritic cells using safe acidotropic drugs may ameliorate autoimmune disease.
  • T cell determinant structure cores and determinant envelopes in three mouse major histocompatibility complex haplotypes . J-Exp-Med 173:609-17.
  • BSF- 2/IL-6 The essential role of B cell stimulatory factor 2 (BSF- 2/IL-6) for the terminal differentiation of B cells. J-Exp-Med 167:332-44.

Landscapes

  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Cell Biology (AREA)
  • Chemical & Material Sciences (AREA)
  • General Health & Medical Sciences (AREA)
  • Immunology (AREA)
  • Public Health (AREA)
  • Animal Behavior & Ethology (AREA)
  • Veterinary Medicine (AREA)
  • Pharmacology & Pharmacy (AREA)
  • Medicinal Chemistry (AREA)
  • Epidemiology (AREA)
  • Engineering & Computer Science (AREA)
  • Microbiology (AREA)
  • Mycology (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Organic Chemistry (AREA)
  • Biomedical Technology (AREA)
  • Zoology (AREA)
  • Wood Science & Technology (AREA)
  • Genetics & Genomics (AREA)
  • Biotechnology (AREA)
  • Biochemistry (AREA)
  • Gastroenterology & Hepatology (AREA)
  • Hematology (AREA)
  • Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
  • General Chemical & Material Sciences (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • General Engineering & Computer Science (AREA)
  • Oncology (AREA)
  • Communicable Diseases (AREA)
  • Medicines Containing Antibodies Or Antigens For Use As Internal Diagnostic Agents (AREA)
  • Medicines Containing Material From Animals Or Micro-Organisms (AREA)
  • Micro-Organisms Or Cultivation Processes Thereof (AREA)

Abstract

Il a été démontré que des substances provoquant une acidification de l'endosome précoce de cellules dendritiques font que ces cellules sont porteuses de davantage d'épitopes qu'elles n'en auraient en l'absence desdites substances. Cette invention porte donc sur l'utilisation de ces substances dans le cadre du traitement d'infection chroniques établies et de cancers. Elle a également trait à des vaccins, destinés au traitement de ces infections chroniques établies ou de cancers, faisant appel à ces substances et/ou aux cellules dendritiques susmentionnées et/ou à des antigènes spécifiques de ces infections chroniques établies et cancers.
PCT/GB1998/003583 1997-12-01 1998-12-01 Traitement d'infections chroniques etablies et de cancers WO1999027955A2 (fr)

Priority Applications (1)

Application Number Priority Date Filing Date Title
AU13432/99A AU1343299A (en) 1997-12-01 1998-12-01 Treatment of established chronic infections and cancers

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
GB9725480.9 1997-12-01
GBGB9725480.9A GB9725480D0 (en) 1997-12-01 1997-12-01 Cytokines and their use in the treatment of established chronic infections and cancers

Publications (2)

Publication Number Publication Date
WO1999027955A2 true WO1999027955A2 (fr) 1999-06-10
WO1999027955A3 WO1999027955A3 (fr) 1999-08-12

Family

ID=10822983

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/GB1998/003583 WO1999027955A2 (fr) 1997-12-01 1998-12-01 Traitement d'infections chroniques etablies et de cancers

Country Status (3)

Country Link
AU (1) AU1343299A (fr)
GB (1) GB9725480D0 (fr)
WO (1) WO1999027955A2 (fr)

Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP0241725A2 (fr) * 1986-03-17 1987-10-21 BOEHRINGER INGELHEIM INTERNATIONAL GmbH Vaccins
WO1995006077A2 (fr) * 1993-08-27 1995-03-02 Sandoz Ltd. Matrices polymeres et leurs utilisations dans des compositins pharmaceutiques
DE19535853A1 (de) * 1995-09-18 1997-03-20 Fraunhofer Ges Forschung Varianten des humanen rekombinanten Interferon-gamma (rhu-IFN-gamma) mit erhöhter thermischer Stabilität
WO1997028808A1 (fr) * 1996-02-12 1997-08-14 The Scripps Research Institute Procedes d'inhibition de l'exportation de proteines sans sequence de tete a l'aide de glycosides ou aglycones cardiaques et de derives

Patent Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP0241725A2 (fr) * 1986-03-17 1987-10-21 BOEHRINGER INGELHEIM INTERNATIONAL GmbH Vaccins
WO1995006077A2 (fr) * 1993-08-27 1995-03-02 Sandoz Ltd. Matrices polymeres et leurs utilisations dans des compositins pharmaceutiques
DE19535853A1 (de) * 1995-09-18 1997-03-20 Fraunhofer Ges Forschung Varianten des humanen rekombinanten Interferon-gamma (rhu-IFN-gamma) mit erhöhter thermischer Stabilität
WO1997028808A1 (fr) * 1996-02-12 1997-08-14 The Scripps Research Institute Procedes d'inhibition de l'exportation de proteines sans sequence de tete a l'aide de glycosides ou aglycones cardiaques et de derives

Non-Patent Citations (3)

* Cited by examiner, † Cited by third party
Title
CHAIN, B. M. ET AL: "Processing and presentation by dendritic cells. The role of th lysosome in antigen breakdown" REGUL. IMMUNE GENE EXPRESSION, ÄPROC. CONF.Ü (1986), MEETING DATE 1985, 207-19. EDITOR(S): FELDMANN, MARC;MCMICHAEL, ANDREW J. PUBLISHER: HUMANA, CLIFTON, N. J. CODEN: 55LJAC, XP002096617 *
DRAKESMITH H ET AL: "In vivo priming of T cells against cryptic determinants by dendritic cells exposed to interleukin 6 and native antigen." PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA, (1998 DEC 8) 95 (25) 14903-8. JOURNAL CODE: PV3. ISSN: 0027-8424., XP002096616 United States *
LUTZ ET AL.: "Intracellular routes and selective retention of antigens in mildly acidic cathepsin D/lysosome-associated membrane protein-1/MHC Class II-positive vesicles in immature dendritic cells" THE JOURNAL OF IMMUNOLOGY, vol. 159, 1997, pages 3707-3716, XP002096615 cited in the application *

Also Published As

Publication number Publication date
AU1343299A (en) 1999-06-16
WO1999027955A3 (fr) 1999-08-12
GB9725480D0 (en) 1998-01-28

Similar Documents

Publication Publication Date Title
Su et al. Vaccination against chlamydial genital tract infection after immunization with dendritic cells pulsed ex vivo with nonviable Chlamydiae
Muir et al. Insulin immunization of nonobese diabetic mice induces a protective insulitis characterized by diminished intraislet interferon-gamma transcription.
JP2547162B2 (ja) 哺乳動物における異系移植片拒否反応を抑制するための方法と組成物
JP2005528091A (ja) Ｔ細胞由来小胞の機能化、および免疫原性医薬組成物を製造するためのその使用
CA2063416C (fr) Methodes de traitement et de prevention de l'uveoretinie chez les mammiferes
Xiao et al. Mechanisms of recovery from experimental allergic encephalomyelitis induced with myelin basic protein peptide 68–86 in Lewis rats: a role for dendritic cells in inducing apoptosis of CD4+ T cells
JP3761574B2 (ja) Ｔ細胞活性化
Ramírez et al. Induction of resistance to active experimental allergic encephalomyelitis by myelin basic protein‐specific Th2 cell lines generated in the presence of glucocorticoids and IL‐4
EP0964697B1 (fr) Applications therapeutiques d'antigenes ou d'epitopes associes a un traitement peptidique cellulaire altere, exprimes par exemple sur des cellules rma-s transfectees avec un gene b7-1
Matsumoto et al. Limiting-dilution analysis of the frequency of myelin basic protein-reactive T cells in Lewis, PVG/c and BN rats. Implication for susceptibility to autoimmune encephalomyelitis.
Watanabe et al. Experimental autoimmune thyroiditis induced by thyroglobulin-pulsed dendritic cells
US5733550A (en) Method for enhancing the association of exogenous peptides with class I MHC molecules on the surface of antigen presenting cells with β-microglobulin
WO1999027955A2 (fr) Traitement d'infections chroniques etablies et de cancers
AU6042894A (en) Soluble T-cell receptor alpha chain and derivatives used as prophylactic and therapeutic agents for autoimmune diseases
US20230201323A1 (en) Vaccine formulations
WO1991008301A1 (fr) PROTEINES EMPECHANT L'INTERACTION ENTRE UN FRAGMENT Fc D'UNE IMMUNOGLOBULINE ET SON RECEPTEUR ET UTILISATION EN THERAPEUTIQUE, NOTAMMENT DANS LE TRAITEMENT DES AFFECTIONS LIEES AUX VIRUS HIV
US20130337001A1 (en) Immunogenic composition for treatment of hepatitis C, a method for preparing the composition and use thereof for treating hepatitis C
US20230340416A1 (en) Augmentation of fibroblast therapeutic activity by complement blockade and/or inhibition
IL256199B1 (en) A preparation containing a combination of peptides
WO1999021968A1 (fr) Immunite cellulaire inductible par l'activation de reactions dependantes de th1 et la suppression de reactions dependantes de th2 au moyen de macrophages
Seddon et al. Deaggregated homologous immunoglobulin‐peptide conjugates induce peptide‐specific T cell nonresponsiveness in vivo
RU2420311C2 (ru) Вакцина на основе в-клеток, нагруженных лигандом т-клеток-природных киллеров и антигеном
WO1998035696A1 (fr) Utilisation de thymosine alpha 1 pour la fabrication d'un medicament destine a favoriser le developpement de cellules souches
CpG-C Local and Systemic Effects of Intranodally
Mueller et al. Cloning of dendritic cell-expressed genes

Legal Events

Date Code Title Description
AK Designated states

Kind code of ref document: A2

Designated state(s): AL AM AT AU AZ BA BB BG BR BY CA CH CN CU CZ DE DK EE ES FI GB GD GE GH GM HR HU ID IL IS JP KE KG KP KR KZ LC LK LR LS LT LU LV MD MG MK MN MW MX NO NZ PL PT RO RU SD SE SG SI SK SL TJ TM TR TT UA UG US UZ VN YU ZW

AL Designated countries for regional patents

Kind code of ref document: A2

Designated state(s): GH GM KE LS MW SD SZ UG ZW AM AZ BY KG KZ MD RU TJ TM AT BE CH CY DE DK ES FI FR GB GR IE IT LU MC NL PT SE BF BJ CF CG CI CM GA GN GW ML MR NE SN TD TG

121 Ep: the epo has been informed by wipo that ep was designated in this application
AK Designated states

Kind code of ref document: A3

Designated state(s): AL AM AT AU AZ BA BB BG BR BY CA CH CN CU CZ DE DK EE ES FI GB GD GE GH GM HR HU ID IL IS JP KE KG KP KR KZ LC LK LR LS LT LU LV MD MG MK MN MW MX NO NZ PL PT RO RU SD SE SG SI SK SL TJ TM TR TT UA UG US UZ VN YU ZW

AL Designated countries for regional patents

Kind code of ref document: A3

Designated state(s): GH GM KE LS MW SD SZ UG ZW AM AZ BY KG KZ MD RU TJ TM AT BE CH CY DE DK ES FI FR GB GR IE IT LU MC NL PT SE BF BJ CF CG CI CM GA GN GW ML MR NE SN TD TG

DFPE Request for preliminary examination filed prior to expiration of 19th month from priority date (pct application filed before 20040101)
NENP Non-entry into the national phase

Ref country code: KR

REG Reference to national code

Ref country code: DE

Ref legal event code: 8642

122 Ep: pct application non-entry in european phase
NENP Non-entry into the national phase

Ref country code: CA